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Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated
Sue Ping Lim1, Raman Kumar, Yamini Akkamsetty
1Cancer Therapeutics Laboratory, Centre for Personalized Cancer Medicine, The University of Adelaide, Adelaide, South Australia, Australia. sue.lim@adelaide.edu.au
Background:
Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation.
Methods:
A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system.
Results:
Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation.
Conclusions:
The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
Insights
A new cell-based assay, EPISSAY, enables rapid screening of gene reactivation for epigenetic drugs. This system demonstrated that PEGylated liposomal decitabine has 50% higher potency than the free drug.
Area of Science:
- Epigenetics
- Drug Discovery
- Bioassay Development
Background:
- Assessing epigenetic drug efficacy requires rapid screening bioassays.
- Gene reactivation is a key indicator of epigenetic drug activity.
- Current methods for assessing epigenetic drug activity are limited.
Purpose of the Study:
- To develop a novel cell-based assay system for screening gene reactivation.
- To validate the EPISSAY system using known epigenetic drugs and drug formulations.
- To compare the potency of free decitabine with PEGylated liposomal decitabine.
Main Methods:
- Developed EPISSAY using a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) in MCF10A cells.
- Validated EPISSAY with the target gene TXNIP and decitabine formulated in PEGylated liposomes.
- Measured RFP expression changes following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors.
Main Results:
- EPISSAY successfully detected gene reactivation indicated by increased RFP expression upon treatment with decitabine and vorinostat.
- The assay quantified a 50% increase in potency for decitabine encapsulated in PEGylated liposomes compared to free decitabine.
- PEGylated liposomal encapsulation protected decitabine from rapid degradation, enhancing its efficacy.
Conclusions:
- EPISSAY is a novel, rapid bioassay system for evaluating gene reactivation.
- The system allows for efficient comparison of existing and novel epigenetic drugs.
- EPISSAY facilitates the assessment of drug formulations, such as liposomal encapsulation, for improved epigenetic drug delivery and efficacy.
